Herpes simplex viruses infect humans worldwide. In the US, herpes simplex virus type 2 (HSV-2) infects about 17% of adults. Rates are far higher in the developing world, especially in populations with high HIV-1 prevalence. HSV-2 infections are permanent and the virus establishes latency in dorsal root ganglia neurons after infection. Periodic reactivation leads to HSV-2 shedding, although transmitting persons usually do not have symptoms or lesions when they transmit virus. HSV-2 has serious medical consequences including fatal/disabling neonatal infection and infection renders persons twice as susceptible to HIV-1 infection. Therapeutic manipulation of the immune response (immunotherapy) is a good strategy to impact symptomatic disease, HSV-2 shedding, and ultimately, HSV-2 transmission in the community. Vaccine trials and non-clinical human and animal studies show that cellular immunity controls the duration and severity of HSV lesions. HSV-specific CD8+ T cells localize to infected ganglia in humans and CD8+ T cell presence correlates with viral clearance. HSV-2 tegument proteins, including those encoded by genes UL46 and UL47 are prominent CD8+ (and CD4+) T cell antigens in humans. We propose that boosting HSV-2-specific CD8+ CTL in infected humans will have a therapeutic effect. Our previous STTR Phase I data show that HSV-2 tegument immunogens can be manufactured and formulated as plasmid DNA (pDNA) vaccines encoding viral proteins. The pDNA vaccines are well tolerated, elicit CD8+ T-cell responses in mice, and are protective in a murine intravaginal lethal challenge model. We also confirm work of others that truncated gD2 pDNA vaccine is immunogenic and protective. The overall goal of this phase II STTR is to carry out non-clinical animal efficacy and safety studies in support of an IND to allow rapid translation of the vaccine to the clinic. This Phase II proposal, as per NIH policy, starts six or less submission dates after Phase I. We propose the following Specific Aims: Aim 1. Determine the optimum combination of UL46, UL47, and gD2 vaccines for elicitation of HSV-2- specific CD8+ T-cell responses. Determine dose and combinations with clinical activity in two murine models with endpoints being acute intravaginal challenge and flank zosteriform spread. Aim 2. Determine the activity of an optimized UL46/UL47/gD2 vaccine for reduction of recurrent vaginal HSV-2 shedding in the guinea pig therapeutic model. Aim 3. Compare the immunogenicity and therapeutic efficacy of electroporation-assisted and intramuscular immunization with HSV-2 pDNA vaccines in the murine and guinea pig models. Aim 4. Based on results from Aims 1-3, plan and initiate non-clinical safety studies on a candidate pDNA HSV-2 immunotherapeutic vaccine and file an IND with the FDA.